Certain endogenous steroids and their synthetic analogues (neuroactive steroids) produce profound and rapid effects on the central nervous system ranging from general anesthesia to seizures. While these effects are thought to result from steroid interactions with specific binding sites of the GABAA receptor, methods have not been available to identify and characterize these putative neuroactive steroid binding sites. This proposal is based on the development, by the applicant lab, of a photoaffinity labeling reagent (PAL-1) for the putative neuroactive steroid binding site(s). PAL-1 has been shown to modulate GABA-activated chloride currents at concentrations less than 5 nanomolar, and to have appropriate photochemical properties to function as an efficient photolabeling reagent. This project will use PAL-1 to achieve three specific goals: (10 The proteins and proteins subunits that have specific binding sites for neuroactive steroids will be identified, and the stoichiometry and affinity of steroid binding will be measured. To accomplish this goal, a radioactive analogue of PAL-1 will be synthesized and photolabeling experiments will be performed on purified GABAA receptors and on brain tissue; labeled proteins will be identified by protein electrophoresis, measurement of protein incorporation of radioactivity and immunoblotting. (2) To determine the relationship between neuroactive steroid binding sites and the sites of action of other neuroactive drugs, studies will be performed examining the effects of various anesthetics, sedatives, convulsants and anti-convulsants on PAL-1 labeling in brain tissue. (3) To determine the amino acid sequences labeled by PAL-1, photolabeling studies will also be performed on cultured cells transfected with specific combinations of GABAA receptor subunits. Labeled peptides will be purified by immunoprecipitation and microsequenced. The information gained from this project will provide the background knowledge and pharmacologic tools to: 1) approach the question of how endogenous neuroactive steroids modulate CNS function in health and disease and; 2) to develop new pharmaceutical agents including potent steroidal anesthetics with minimal side effects, novel anticonvulsants and neuroactive steroid antagonists.